This project involves evaluating common human and animal paramyxoviruses as potential human vaccine vectors against highly pathogenic viruses. We previously evaluated human parainfluenza virus type 3 (HPIV3) as a vector to express the spike glycoprotein of Severe Acute Respiratory Syndrome Coronavirus (SARS) from an added gene. A single dose of the HPIV3-S construct administered by the combined intranasal (IN) and intratracheal (IT) routes was immunogenic and protective against SARS challenge in African green monkeys (AGM). We also evaluated HPIV3 expressing the single glycoprotein GP of Ebola virus (EBOV). A single respiratory tract inoculation was completely protective against EBOV in guinea pigs and protected 88% of rhesus monkeys against EBOV challenge;two doses provided complete protection. HPIV3 is a common human pathogen and essentially all adults have a history of natural infection with HPIV3. Therefore, it was important to determine whether previous infection with HPIV3 would restrict the replication and immunogenicity of the HPIV3 vector. We previously found that, in guinea pigs that were infected with HPIV3 and challenged 40 days later with HPIV3/EboGP, replication of the vector could not be detected, indicating a high level of restriction. Surprisingly, however, the immune response to EBOV GP was almost equivalent to that achieved in control animals that had not been previously infected with HPIV3. In the experiments continuing into present year, rhesus monkeys were infected twice with HPIV3 and, 11 months following the second infection, were immunized with two doses of HPIV3/EboGP given 4 weeks apart. After a single dose of HPIV3/EboGP, the titers of EBOV-specific serum ELISA or neutralization antibodies were substantially less in HPIV3-immune animals compared to HPIV3-naive animals. However, after two doses, which were previously determined to be required for complete protection against EBOV challenge, the antibody titers were indistinguishable between the two groups. The vaccine virus appeared to replicate, at a reduced level, in the respiratory tract despite the preexisting immunity. This may reflect the known ability of HPIV3 to re-infect and may also reflect the presence of EBOV GP in the vector virion, which confers resistance to neutralization in vitro by HPIV3-specific antibodies. Thus, an HPIV3-based vector was substantially immunogenic even in the face of strong pre-existing immunity to the vector. In another construct, we deleted the HPIV3 F and HN genes in HPIV3-GP, leaving the foreign GP as the sole viral surface protein. This virus was highly attenuated in guinea pigs, but retained its immunogenicity and was completely protective against EBOV challenge. We are presently arranging to have this construct evaluated for immunogenicity and efficacy in rhesus monkeys. All of these vectors appeared to remain highly restricted to the respiratory tract and were not significantly immunogenic when delivered parenterally. We also have been investigating the use of the avian Newcastle disease virus (NDV) as a human vaccine vector. NDV is antigenically distinct from common human pathogens and should not be affected by pre-existing immunity. There is anecdotal evidence that NDV is highly restricted in humans and does not cause significant disease. Previously, we confirmed that NDV is very highly attenuated following IN/IT inoculation of rhesus monkeys and AGM, and found that expressed foreign proteins are moderately-to-highly immunogenic. In AGM, two doses of NDV expressing the SARS S protein were strongly immunogenic and protective against challenge with a high dose of SARS. In studies continuing into the present year, we engineered Newcastle disease virus (NDV), an avian paramyxovirus, as a vector to separately express the HPAIV hemagglutinin (HA) protein, or a second version in which the polybasic cleavage site was replaced with that from a low-pathogenicity strain (HA/RV), or the HPAIV neuraminidase (NA). The three vaccine viruses NDV/HA, NDV/HA/RV, and NDV/NA were administered separately to groups of AGMs by the intranasal/intratracheal route. An additional group of animals received NDV/HA by aerosol administration. Each of the vaccine constructs was highly restricted for replication, with only low levels of virus shedding detected in respiratory secretions. All groups developed high levels of neutralizing antibodies against homologous and heterologous strains of HPAIV and were protected against challenge with 2 x 10(7) PFU of homologous HPAIV. The high level of restriction of HPAIV challenge virus was established by assay of nasal swabs and tracheal lavages for virus by infectious virus assay and RT-PCR, by direct assay for infectious virus in harvested tissue, by immunohistochemical analysis of harvested tissue, and by profiling challenge-induced pulmonary host gene expression. The finding that the NA protein also was highly immunogenic and protective was somewhat surprising, since the NA protein had not been considered to be a potent neutralization or protective antigen. These results showed that the modified HA gene and the NA gene are the genes of choice for inclusion in a vectored vaccine for human use. This study also successfully evaluated the feasibility of aerosol delivery of NDV-vectored vaccines. NDV represents serotype 1 of the avian paramyxoviruses (APMV). There are 8 other serotypes, namely APMV-2 to -9. There is a great deal of information available for NDV because of its importance in poultry. In contrast, relatively little was known about the other serotypes, apart from a complete sequence for one strain of APMV-6. Working with collaborators at the University of Maryland at College Park, we previously initiated antigenic and sequence analysis of these as a prelude to their evaluation for safety, replication, and immunogenicity in non-human primates as potential vectors. In studies continuing in the present year, we have determined complete consensus sequences for all of the serotypes, and in a number of instances have sequences for two to four different strains for each serotype. This has provided evidence for antigenic and sequence heterogeneity within serotypes. The antigenic relationships between the serotypes were previously determined mostly by hemagglutination-inhibition assays, which thus provide information only for a limited part of a single glycoprotein. These relationships are being re-evaluated using polyvalent post-infection sera and using neutralization assays. In addition, these serotypes are now being evaluated for replication and pathogenicity in chicken eggs, chickens, turkeys, hamsters, and mice, in preparation for evaluation in non-human primates.